PROJECT SUMMARY Breast cancer has become the predominant cancer among women and despite the dramatic improvement in prevention and early diagnosis;it is still the second leading cause of cancer death among women. A substantial number of breast cancer patients present metastases, a therapeutic capable of destroying them, either alone or in combination with other therapies, would be of substantial benefit. The ideal therapeutic agent must be non-toxic, non-immunogenic, should replicate throughout the tumor and can be easily removed from the body after therapy. Salmonella enterica serovar Typhimurium, a facultative anaerobic bacterium that infects a wide variety of animal hosts, naturally accumulates in most solid murine tumors versus normal murine tissues at a ratio of 1000:1 [1]. Avirulent Salmonella mutant strains have been used to directly kill tumors (Zhao et al., 05, 06) or to deliver constitutively expressed therapeutic proteins to tumors in mouse models (Avogadri et al., 2005; Bermudes et al., 2001;King et al., 2002;Mei et al., 2002;Loeffler et al., 2007). Avirulent Salmonella mutants used in cancer therapy were generally selected for their avirulence not their tumor targeting ability. For example, the Salmonella strain VNP20009, commonly use in cancer research, showed high safety level but only a moderate tumor targeting when tested in phase I human clinical trials in patients with metastatic melanoma (Toso et al., 2002, Cunningham et al., 2001). Our first goal is to optimize Salmonella as a therapeutic delivery system, to accomplish this goal we propose to test the effect of mutations in every non- essential Salmonella gene and measure their effect on virulence and tumor targeting. We previously developed the technology of high-throughput screening of Salmonella transposon libraries in tumors. Salmonella promoters typically used to drive the therapeutic genes are constitutively expressed. However, the tumor environment (e.g., hypoxia, acidic conditions) is likely to result in activation of certain Salmonella promoters in the tumor, but not in other healthy and well-oxygenated tissues (Mengesha et al., '06). Therefore, we anticipate that the use of endogenous Salmonella-based tumor-specific promoters we previously identified [Arrach et, al. 2008], will further increase the specificity and reduce toxic side effects of drug therapy. So far, no comprehensive survey has been conducted to identify such "tumor-specific" promoters. In fact, we were the only group to publish the first high-throughput method on bacterial promoter activation in tumors (Arrach et al., 2008). Our second goal is to identify and dissect the regulatory aspect of Salmonella promoters that are preferentially induced in 4T1 orthotopic syngeneic breast tumors. Our long term goal is to combine selected mutants from Aim 1 with promoter candidates from Aim 2 to express therapeutic proteins in human breast xenografts from pristine patients. Such combination may lead to synergistic improvements in tumor killing, while simultaneously reducing the risk of damage to the normal tissue